There is an urgent need for novel prophylactic methods, such as topical microbicides, designed for genital or rectal application to prevent infection with HIV and other sexually transmitted pathogens. The overall goal of this Program is to develop safe and effective combinations of topical microbicides for vaginal or rectal use to prevent transmission of HIV and other sexually transmitted diseases. PRO 2000, a naphthalene sulfonic acid, is active against wide range of HIV isolates in vitro and also inhibits herpes simplex virus (HSV) infection in vitro and in animal models. A combination of PRO 2000 with a second microbicide that targets a different step in the HIV life cycle might have advantages over either drug alone including an expanded spectrum of activity, enhanced efficacy and possibly reduced mucosal toxicity. The focus of this Program, therefore, is to identify optimal combinations of candidate topical microbicides that would act additively or synergistically with PRO 2000 and provide even greater protection. An iterative approach toward identifying the best combinations of topical microbicides with an excellent safety profile is proposed. Project 1 will evaluate candidate combinations against HIV in primary culture systems. Project 2 focuses on evaluating the activity of candidate combinations in biologically relevant culture systems against HSV-2, a major co-factor in HIV transmission, alone and in combination with HIV. Candidate combinations will be evaluated using a three-tier approach. In Specific Aim 1, combinations will be evaluated for anti-HSV activity and safety using human cervical cultures in the presence of cervicovaginal secretions and semen. Leading combinations will then be evaluated for their impact on mucosal immunity and efficacy in a dual infection model of HSV and HIV using human explant cultures (cervical, vaginal and rectal) in Specific Aim 2. The combinations with the greatest efficacy and safety profile will be evaluated in murine genital and rectal models for cytotoxicity, inflammatory effects, and efficacy against HSV. Results in animal models will be correlated with those obtained for PRO 2000 in pilot human trials in Project 3. The efficacy and safety data obtained in these relevant biological cultures and animal models may provide compelling support for advancing a leading combination to clinical trials.